In Vitro Evaluation of Extracellular Hydrolytic Cell Wall Degrading Enzymes of Fusarium oxysporum f. sp. elaeidis, Pathogen of Oil Palm Vascular Wilt

The oil palm, Elaeis guineensis Jacq, remains the world's most productive source of vegetable oil, yielding far more oil per hectare than any other oil crop (Hartley, 1988; Oil World, 2008). Production is nonetheless constrained by several diseases, of which vascular wilt caused by the soil-borne fungus Fusarium oxysporum f. sp. elaeidis (Foe) is the most economically damaging in West and Central Africa, where losses of 25–50% of a plantation are not unusual (Renard and De Franqueville, 1989; Flood, 2006). Foe colonizes the root and vascular system, and disease severity is closely tied to the balance between the pathogen's invasive capacity and the host's ability to wall off infected xylem tissue (Corley and Tinker, 2003; Cooper, 2011). Selection of tolerant planting material remains the most effective control measure currently available (Renard and De Franqueville, 1989), which makes a clearer understanding of how the pathogen actually breaches host tissue directly relevant to breeding and screening programs.

Central to that invasive capacity is the fungus's ability to secrete extracellular cell wall-degrading enzymes (CWDEs). The plant cell wall, built mainly from cellulose, hemicellulose, pectin and lignin, is the first physical barrier a pathogen must overcome, and necrotrophic and hemibiotrophic fungi typically breach it by secreting a battery of hydrolytic enzymes that loosen and macerate the wall (Bateman and Millar, 1966; Carpita and Gibeaut, 1993; Annis and Goodwin, 1997). Cellulose is broken down synergistically by endoglucanases, exoglucanases (cellobiohydrolases) and β-glucosidases (Henrissat, 1994; Dashtban et al., 2009; Maki et al., 2009), while pectin is degraded by pectinases, which are usually produced earliest and in the largest amount because they alone are capable of macerating living plant tissue (Carpita and Gibeaut, 1993; Annis and Goodwin, 1997). The amount and mix of enzymes a given isolate secretes are not fixed: they respond to the available carbon source, incubation time, pH and temperature, and this physiological plasticity is thought to contribute to differences in aggressiveness between isolates (Naessens and Vandamme, 2003).

Foe has previously been shown to produce pectin esterase, polygalacturonase and Cx-cellulase (Osagie and Obuekwe, 1991), but comparative data on the four principal hydrolases examined here—endoglucanase, exoglucanase, β-glucosidase and pectinase—across geographically distinct isolates, and on how their production responds to culture conditions, remain limited. Reducing sugar assays such as the dinitrosalicylic acid (DNS) method (Miller, 1959) and the O-toluidine method for glucose remain the most widely used tools for quantifying this activity (Alexander et al., 2011), and were adopted here for consistency with earlier work on Foe enzymology (Osagie and Obuekwe, 1991).

This study therefore set out to (i) determine the extracellular hydrolytic enzymes endoglucanase, exoglucanase, β-glucosidase and pectinase (endo-polymethylgalacturonase) produced by four pathogenic strains of Foe from Nigeria, Ghana and Cameroon; (ii) establish the culture conditions (carbon source, incubation period, pH and temperature) that optimize production of each enzyme; and (iii) quantify the amount of each enzyme produced under these conditions.